The present invention relates to an improved microcantilever detector and a microcantilever array, and more particularly to an uncoated microcantilever detector used in high throughput detection applications, particularly for detecting hybridization interactions as well as other chemical and biochemical interactions.
Microcantilevers have been demonstrated to be extremely sensitive chemical and biological sensors. The microcantilever resonance frequency changes due to adsorption-induced mass loading. The cantilever also undergoes bending due to adsorption-induced surface stress variation if the adsorption is confined to a single surface. The superior sensitivity of the microcantilever is due to its extremely small mass. In U.S. Pat. No. 5,445,008, Wachter and Thundat describe a microcantilever sensor based on variations in adsorbed mass.
One important disadvantage of a microcantilever sensor is the inability to distinguish between different chemical species. This disadvantage also exists on other mass sensors such as the quartz crystal microbalance (QCM), surface acoustic wave device (SAW), plate wave resonator, and the Lamb and Love wave sensor.
To overcome the disadvantage of lack of chemical selectivity, cantilevers are often-coated with a chemically selective layer. However, this chemically selective layer does not provide absolute chemical selectivity except in the case of biosensors based on antibody-antigen interaction or DNA hybridization. This disadvantage can be overcome by using pattern recognition and orthogonal arrays where each element in the array is coated with a specific chemically selective coating that provides a unique signal.
One important advantage of microcantilever sensors lies in the ability to be arranged into an array for detection of a large number of analytes using a single chip since microcantilevers are very small and a large number of sensor elements can be micro-machined on a single chip. However, one of the main disadvantages of the microcantilever technology is that in an array format, every element in the array must be modified by a different chemical species. This is a very challenging task since the surface area of the cantilever is very small. In addition, when a large number of analytes are present, it is necessary to use pattern recognition techniques for identifying analytes.
However, when only xe2x80x9cyesxe2x80x9d or xe2x80x9cnoxe2x80x9d answers are involved, using a microcantilever as a chemically selective analytical tool is redundant and costly. First and foremost, it is extremely difficult and time consuming to modify every single microcantilever in the array with a discrete chemically selective layer. Secondly, the quality of the deposited film cannot be guaranteed. Thirdly, signal processing from a large number of sensor elements is extremely expensive and involves development of algorithms that can handle multiple input including reference signals. For example, the cantilever response (bending and frequency change) varies with physical parameters such as pressure, temperature, flow rate, pH, presence of ions in the solutions, and other parameters. Relative humidity plays a major role when microcantilevers are used in air.
In the past, the use of the microcantilever detector concept, especially in array applications, has been costly and cumbersome. In recent years, great advances have been made in array technology. For example, many commercially available array chips such as the Affimetrix chip exist. In conventional chips, such as DNA chips, areas as small as 5 xcexcm by 5 xcexcm are modified with definite sequences of single-stranded DNA. These areas form a well-defined array. When a target sequence is introduced, they hybridize with a complimentary sequence in a particular area. In general, targets are tagged with a fluorescent marker, which when exposed to an appropriate wavelength, produces a particular color. This technology, though widely used, suffers from lack of simplicity in reading due to the multiple steps involved.
In response to the need for further research, improved microcantilever apparatuses, microcantilever array and methods of use have been developed. The apparatuses, array and methods disclosed herein are extremely useful for high throughput operations.
Accordingly, objects of the present invention include apparatus and methods for an improved uncoated microcantilever detector, particularly, an uncoated microcantilever detector and microcantilever detector array wherein the sample sites are placed on a separate semi-conducting or conducting substrate and the microcantilever(s) measures the changes before and after chemical/biochemical interaction or hybridization of the sites. Further and other objects of the present invention will become apparent from the description contained herein.
In accordance with one aspect of the present invention, the foregoing and other objects are achieved by an uncoated microcantilever detector apparatus which comprises at least a first microcantilever element, the microcantilever element being uncoated and comprising a material selected from the group consisting of electrically conductive materials and electrically semi-conductive materials; a substrate disposed relative to the first microcantilever element with a known, controlled gap therebetween, the substrate comprising a material selected from the group consisting of electrically conductive materials and electrically semi-conductive materials, the substrate further comprising means for attaching at least one sample upon the substrate proximate to the microcantilever element; a vibration detection means for detecting vibration of the first microcantilever element and providing vibration data; alternating voltage means disposed and connected for imposing an alternating voltage electrical signal to the substrate with respect to the first microcantilever element to induce vibration in the first microcantilever element; instrumentation means disposed and in communication with the first microcantilever element for receiving the vibration data and to determine frequency and amplitude of vibration of the first microcantilever element and for sensing and quantifying the alternating voltage electrical signal applied to the substrate with respect to the first microcantilever element, and further for detecting and quantifying differences in phase angle between the signal applied by the alternating voltage means and the signal generated by the vibration detection means; and the first microcantilever element, the substrate, the vibration detection means, the alternating voltage means, and the instrumentation means being configured to permit a test fluid to pass between the first microcantilever element and the substrate so that a chemical interaction may occur between a component of the test fluid and a component of the sample.
In accordance with a second aspect of the present invention, a method for detecting a component-capable of chemical interaction or hybridization in a fluid test sample comprises the steps of: providing an uncoated microcantilever detector apparatus which comprises at least one microcantilever element, the microcantilever element being uncoated and comprising a material selected from the group consisting of electrically conductive materials and electrically semi-conductive materials, also comprising a substrate positioned adjacent the microcantilever element and disposed relative to the microcantilever element with a known, controlled gap therebetween, the substrate comprising a material selected from the group consisting of electrically conductive materials and electrically semi-conductive materials, the substrate further comprising means for attaching at least one sample upon the substrate proximate to the microcantilever element, further comprising a vibration detection means for detecting vibration of the microcantilever element and providing vibration data, alternating voltage means disposed and connected for imposing an alternating voltage electrical signal to the substrate with respect to the microcantilever element to induce vibration in the microcantilever element, an instrumentation means disposed and connected for receiving vibration data and to determine frequency and amplitude of vibration of the microcantilever element and for sensing and quantifying the alternating voltage electrical signal applied to the substrate with respect to the microcantilever element, and also for detecting and quantifying differences in phase angle between the signal applied by the alternating voltage means and the signal generated by the vibration detection means, the microcantilever element, the substrate, the vibration detection means, the alternating voltage means, and the instrumentation means being configured to permit a test fluid to pass between the microcantilever element and the substrate so that a chemical reaction or hybridization may occur between a component of the test fluid and a component of the sample; causing the test fluid to pass between the microcantilever element and the substrate to allow chemical interaction or hybridization to occur; and determining the frequency and amplitude of vibration of the microcantilever element and quantifying differences in phase angle between the alternating voltage electrical signal applied by the alternating voltage means and signal generated by the vibration detection means to determine whether a chemical interaction or hybridization occurred and to determine the extent of chemical interaction or hybridization which may have occurred between a component of the test fluid and a component of the sample.